This invention relates to the regulation of gene transcription and/or expression. More specifically, this invention relates to polynucleotide regulatory sequences isolated from plants that are capable of initiating and driving the transcription of genes, and the use of such regulatory sequences in the modification of transcription of endogenous and/or heterologous genes.
Gene expression is regulated, in part, by the cellular processes involved in transcription. During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polymerases. Initiation of transcription in eucaryotic cells is regulated by complex interactions between cis-acting DNA motifs, located within the gene to be transcribed, and trans-acting protein factors. Among the cis-acting regulatory regions are sequences of DNA, termed promoters to which RNA polymerase is first bound, either directly or indirectly As used herein, the term xe2x80x9cpromoterxe2x80x9d refers to the 5xe2x80x2 untranslated region of a gene that is associated with transcription and which generally includes a transcription start site. Other cis-acting DNA motifs, such as enhancers, may be situated further up- and/or down-stream from the initiation site.
Both promoters and enhancers are generally composed of several discrete, often redundant, elements each of which may be recognized by one or more trans-acting regulatory proteins, known as transcription factors. Promoters generally comprise both proximal and more distant elements. For example, the so-called TATA box, which is important for the binding of regulatory proteins, is generally found about 25 basepairs upstream from the initiation site. The so-called CAAT box is generally found about 75 basepairs upstream of the initiation site. Promoters generally contain between about 100 and 1000 nucleotides, although longer promoter sequences are possible.
For the development of transgenic plants, constitutive promoters that drive strong transgene expression are preferred. Currently, the only available constitutive plant promoter that is widely used is derived from Cauliflower Mosaic Virus. Furthermore, there exists a need for plant-derived promoters for use in transgenic food plants due to public conceptions regarding the use of viral promoters. Few gymnosperm promoters have been cloned and those derived from angiosperms have been found to function poorly in gymnosperms. There thus remains a need in the art for polynucleotide promoter regions isolated from plants for use in modulating transcription and expression of genes in transgenic plants.
Briefly, isolated polynucleotide regulatory sequences from eucalyptus and pine that are involved in the regulation of gene expression are disclosed, together with methods for the use of such polynucleotide regulatory regions in the modification of expression of endogenous and/or heterologous genes in transgenic plants. In particular, the present invention provides polynucleotide promoter sequences from 5xe2x80x2 untranslated regions of plant genes that initiate and regulate transcription of DNA sequences placed under their control.
In a first aspect, isolated polynucleotide promoter sequences are provided that comprise a DNA sequence selected from the group consisting of: (a) sequences recited in SEQ ID NO: 2-14 and 20; (b) complements of the sequences recited in SEQ ID NO: 2-14 and 20; (c) reverse complements of the sequences recited in SEQ ID NO: 2-14,20; (d) reverse sequences of the sequences recited in SEQ ID NO: 2-14 and 20; and (e) sequences having either 40%, 60%, 75% or 90% identical nucleotides, as defined herein, to a sequence of (a)-(d).
In a related aspect, the present invention provides DNA constructs comprising, in the 5xe2x80x2-3xe2x80x2 direction, a polynucleotide promoter sequence of the present invention, a DNA sequence to be transcribed, and a gene termination sequence. The DNA sequence to be transcribed may comprise an open reading frame of a DNA sequence that encodes a polypeptide of interest or may be a non-coding, or untranslated, region of a DNA sequence of interest. The open reading frame may be orientated in either a sense or antisense direction. Preferably, the gene termination sequence is functional in a host plant. Most preferably, the gene termination sequence is that of the gene of interest, but others generally used in the art, such as the Agrobacterium tumefaciens nopalin synthase terminator may be usefully employed in the present invention. The DNA construct may further include a marker for the identification of transformed cells.
In a further aspect, transgenic plant cells comprising the DNA constructs of the present invention are provided, together with organisms, such as plants, comprising such transgenic cells, and fruits and seeds of such plants.
In yet another aspect, methods for modifying gene expression in a target organism, such as a plant, are provided, such methods including stably incorporating into the genome of the organism a DNA construct of the present invention. In a preferred embodiment, the target organism is a plant, more preferably a woody plant, most preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. 
In another aspect, methods for producing a target organism, such as a plant, having modified gene expression are provided, such methods comprising transforming a plant cell with a DNA construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
In other aspects, methods for identifying a gene responsible for a desired function or phenotype are provided, the methods comprising transforming a plant cell with a DNA construct comprising a polynucleotide promoter sequence of the present invention operably linked to a gene to be tested, cultivating the plant cell under conditions conducive to regeneration and mature plant growth to provide transgenic a plant; and comparing the phenotype of the transgenic plant with the phenotype of non-transformed, or wild-type, plants.
In yet a further aspect, the present invention provides an isolated polynucleotide from Pinus radiata that encodes ubiquitin. In specific embodiments, the isolated polynucleotide comprises a DNA sequence selected from the group consisting of: (a) a sequence recited in SEQ ID NO: 1; (b) complements of the sequence recited in SEQ ID NO: 1; (c) reverse complements of the sequence recited in SEQ ID NO: 1; (d) reverse sequences of the sequence recited in SEQ ID NO: 1; and (e) sequences having either 40%, 60%, 75% or 90% identical nucleotides, as defined herein, to a sequence of (a)-(d). Polypeptides encoded by such polynucleotides are also provided, together with DNA constructs comprising such polynucleotides, and host cells and transgenic organisms, for example plants, transformed with such DNA constructs.
In yet further aspects, the present invention provides isolated polynucleotides comprising the DNA sequence of SEQ ID NO: 21, or a complement, reverse complement or variant of SEQ ID NO: 21, together with DNA constructs comprising such polynucleotides and cells transformed with such sequences. As discussed below, removal of the sequence of SEQ ID NO: 21 from a polynucleotide that comprises the sequence of SEQ ID NO: 21 may enhance expression of the polynucleotide. Conversely, the inclusion of the sequence of SEQ ID NO: 21 in a DNA construct comprising a polynucleotide of interest may decrease expression of the polynucleotide.
The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
The present invention provides isolated polynucleotide regulatory regions that may be employed in the manipulation of plant phenotypes. More specifically, polynucleotide promoter sequences isolated from pine and eucalyptus are disclosed. As discussed above, promoters are components of the cellular xe2x80x9ctranscription apparatusxe2x80x9d and are involved in the regulation of gene expression. Both tissue- and temporal-specific gene expression patterns have been shown to be initiated and controlled by promoters during the natural development of a plant. The isolated polynucleotide promoter sequences of the present invention may thus be employed in the modification of growth and development of plants, and of cellular responses to external stimuli, such as environmental factors and disease pathogens.
Using the methods and materials of the present invention, the amount of a specific polypeptide of interest may be increased or reduced by incorporating additional copies of genes encoding the polypeptide, operably linked to an inventive promoter sequence, into the genome of a target organism, such as a plant. Similarly, an increase or decrease in the amount of the polypeptide may be obtained by transforming the target plant with antisense copies of such genes.
In one embodiment, the present invention provides a polynucleotide sequence isolated from Pinus radiata that encodes a ubiquitin polypeptide. The full-length sequence of this polynucleotide is provided in SEQ ID NO: 1, with the sequence of the promoter region including an intron being provided in SEQ ID NO: 2 and the sequence of the promoter region excluding the intron being provided in SEQ ID NO: 3. In a related embodiment, the present invention provides isolated polypeptides encoded by the isolated polynucleotide of SEQ ID NO: 1.
In further embodiments, the following isolated polynucleotide promoter sequences from Pinus radiata are provided: a cell division control (CDC) gene promoter (SEQ ID NO: 4); a xylogenesis-specific promoter (SEQ ID NO: 5); a 4-coumarate Co-A ligase (4CL) promoter (SEQ ID NO: 6); and a root-specific promoter (SEQ ID NO: 13 and 14). The following isolated polynucleotide promoter sequences from Eucalyptus grandis are also provided: a cellulose synthase promoter (SEQ ID NO: 7-8 and 20); a leaf-specific promoter (SEQ ID NO: 9-11); and an O-methyl transferase (OMT) promoter (SEQ ID NO: 12). Complements of the inventive isolated polynucleotides, reverse complements of such isolated polynucleotides and reverse sequences of such isolated polynucleotides are also provided, together with variants of such sequences. The present invention also encompasses polynucleotide sequences that differ from the disclosed sequences but which, due to the degeneracy of the genetic code, encode a polypeptide which is the same as that encoded by a polynucleotide sequence disclosed herein.
The term xe2x80x9cpolynucleotide(s),xe2x80x9d as used herein, means a single or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised. A polynucleotide may consist of an entire gene, or any portion thereof. Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of xe2x80x9cpolynucleotidexe2x80x9d therefore includes all such operable anti-sense fragments. Anti-sense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson-Benion et al. (1995), Antisense techniques, Methods in Enzymol. 254(23): 363-375 and Kawasaki et al. (1996), Artific. Organs 20 (8): 836-848.
The term xe2x80x9cpolypeptidexe2x80x9d, as used herein, encompasses amino acid chains of any length including full length proteins, wherein amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention may be naturally purified products, or may be produced partially or wholly using recombinant techniques. The term xe2x80x9cpolypeptide encoded by a polynucleotidexe2x80x9d as used herein, includes polypeptides encoded by a nucleotide sequence which includes the partial isolated DNA sequences of the present invention.
All of the polynucleotides and polypeptides described herein are isolated and purified, as those terms are commonly used in the art.
The definition of the terms xe2x80x9ccomplementxe2x80x9d, xe2x80x9creverse complementxe2x80x9d and xe2x80x9creverse sequencexe2x80x9d, as used herein, is best illustrated by the following example. For the sequence 5xe2x80x2 AGGACC 3xe2x80x2, the complement, reverse complement and reverse sequence are as follows:
As used herein, the term xe2x80x9cvariantxe2x80x9d covers any sequence which has at least about 40%, more preferably at least about 60%, more preferably yet at least about 75% and most preferably at least about 90% identical residues (either nucleotides or amino acids) to a sequence of the present invention. The percentage of identical residues is determined by aligning the two sequences to be compared, determining the number of identical residues in the aligned portion, dividing that number by the total length of the inventive, or queried, sequence and multiplying the result by 100.
Polynucleotide or polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another polynucleotide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences arc the BLASTN and EASTA algorithms. The similarity of polypeptide sequences may be examined using the BLASTP algorithm. Both the BLASTN and BLASTP software are available on the NCBI anonymous FTP server. The BLASTN algorithm version 2.0.4 [Feb. 24, 1998], set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN and BLASTP, is described at NCBI""s website and in the publication of Altschul, Stephen F., et al. (1997), xe2x80x9cGapped BLAST and PSI-BLAST: a new generation of protein database search programsxe2x80x9d, Nucleic Acids Res. 25:3389-3402. The computer algorithm FASTA is available on the Internet and, Version 2.u4, February 1996, set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of variants according to the present invention. The use of the FASTA algorithm is described in W. R. Pearson and D. J. Lipman, xe2x80x9cImproved Tools for Biological Sequence Analysis,xe2x80x9d Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988) and W. R. Pearson, xe2x80x9cRapid and Sensitive Sequence Comparison with FASTP and FASTA,xe2x80x9d Methods in Enzymology 183:63-98 (1990).
The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity: Unix running command: blastall -p blastn -d embldb -e 10 -G 1 -E 1 -r 2 -v 50 -b 50 -i queryseq -o results; and parameter default values:
-p Program Name [String]
-d Database [String]
-e Expectation value (E) [Real]
-G Cost to open a gap (zero invokes default behavior) [Integer]
-E Cost to extend a gap (zero invokes default behavior) [Integer]
-r Reward for a nucleotide match (blastn only) [Integer]
-v Number of one-line descriptions (V) [Integer]
-b Number of alignments to show (B) [Integer]
-i Query File [File In]
-o BLAST report Output File [File Out] Optional
For BLASTP the following running parameters are preferred: blastall -p blastp -d swissprotdb -e 10 -G 1 -E 1 -v 50 -b 50 -i queryseq -o results
-p Program Name [String]
-d Database [String]
-e Expectation value (E) [Real]
-G Cost to open a gap (zero invokes default behavior) [Integer]
-E Cost to extend a gap (zero invokes default behavior) [Integer]
-v Number of one-line descriptions (v) [Integer]
-b Number of alignments to show (b) [Integer]
-I Query File [File In]
-o BLAST report Output File [File Out] Optional
The xe2x80x9chitsxe2x80x9d to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
The BLASTN and FASTA algorithms also produce xe2x80x9cExpectxe2x80x9d values for alignments. The Expect value (E) indicates the number of hits one can xe2x80x9cexpectxe2x80x9d to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions of the sequences then have a probability of 90% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.
According to one embodiment, xe2x80x9cvariantxe2x80x9d polynucleotides, with reference to each of the polynucleotides of the present invention, preferably comprise sequences having the same number or fewer nucleic acids than each of the polynucleotides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide of the present invention. That is, a variant polynucleotide is any sequence that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at the default parameters. According to a preferred embodiment, a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at the default parameters.
Variant polynucleotide sequences will generally hybridize to the recited polynucleotide sequence under stringent conditions. As used herein, xe2x80x9cstringent conditionsxe2x80x9d refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65xc2x0 C., 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1X SSC, 0.1% SDS at 65xc2x0 C. and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65xc2x0 C.
As used herein, the term xe2x80x9cx-mer,xe2x80x9d with reference to a specific value of xe2x80x9cx,xe2x80x9d refers to a polynucleotide comprising at least a specified number (xe2x80x9cxxe2x80x9d) of contiguous residues of any of the polynucleotides identified as SEQ ID NO: 1-14 and 20. The value of x may be from about 20 to about 600, depending upon the specific sequence.
Polynucleotides of the present invention comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 1-14 and 20 or their variants. According to preferred embodiments, the value of x is preferably at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides of the present invention include polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer a 250-mer, or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide identified as SEQ ID NO: 1-14 and 20 or a variant of one of the polynucleotides identified as SEQ ID NO: 1-14 and 20.
The inventive polynucleotides may be isolated by high throughput sequencing of cDNA libraries prepared from Eucalyptus grandis and Pinus radiata as described below. Alternatively, oligonucleotide probes based on the sequences provided in SEQ ID NO: 1-14 and 20 can be synthesized and used to identify positive clones in either cDNA or genomic DNA libraries from Eucalyptus grandis and Pinus radiata by means of hybridization or PCR techniques. Probes can be shorter than the sequences provided herein but should be at least about 10, preferably at least about 15 and most preferably at least about 20 nucleotides in length. Hybridization and PCR techniques suitable for use with such oligonucleotide probes are well known in the art, and include those taught by Sambrook et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). Positive clones may be analyzed by restriction enzyme digestion, DNA sequencing and the like.
In addition, the DNA sequences of the present invention may be generated by synthetic means using techniques well known in the art. Equipment for automated synthesis of oligonucleotides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems Division (Foster City, Calif.) and may be operated according to the manufacturer""s instructions.
Polypeptides of the present invention may be prepared recombinantly by inserting a DNA sequence that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells.
As noted above, the inventive polynucleotide promoter sequences may be employed in DNA constructs to drive transcription and/or expression of a DNA sequence of interest. The DNA sequence of interest may be either endogenous or heterologous to the organism, for example plant, to be transformed. The inventive DNA constructs may thus be employed to modulate levels of transcription and/or expression of a DNA sequence, for example gene, that is present in the wild-type plant, or may be employed to provide transcription and/or expression of a DNA sequence that is not found in the wild-type plant.
In certain embodiments, the DNA sequence of interest comprises an open reading frame that encodes a target polypeptide. The open reading frame is inserted in the DNA construct in either a sense or antisense orientation, such that transformation of a target plant with the DNA construct will lead to a change in the amount of polypeptide compared to the wild-type plant. Transformation with a DNA construct comprising an open reading frame in a sense orientation will generally result in over-expression of the selected polypeptide, while transformation with a DNA construct comprising an open reading frame in an antisense orientation will generally result in reduced expression of the selected polypeptide. A population of plants transformed with a DNA construct comprising an open reading frame in either a sense or antisense orientation may be screened for increased or reduced expression of the polypeptide in question using techniques well known to those of skill in the art, and plants having the desired phenotypes may thus be isolated.
Alternatively, expression of a target polypeptide may be inhibited by inserting a portion of the open reading frame, in either sense or antisense orientation, in the DNA construct. Such portions need not be full-length but preferably comprise at least 25 and more preferably at least 50 residues of the open reading frame. A much longer portion or even the full length DNA corresponding to the complete open reading frame may be employed. The portion of the open reading frame does not need to be precisely the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target gene. Thus a sequence derived from one species may be used to inhibit expression of a gene in a different species.
In further embodiments, the inventive DNA constructs comprise a DNA sequence including an untranslated, or non-coding, region of a gene coding for a target polypeptide, or a DNA sequence complementary to such an untranslated region. Examples of untranslated regions which may be usefully employed in such constructs include introns and 5xe2x80x2-untranslated leader sequences. Transformation of a target plant with such a DNA construct may lead to a reduction in the amount of the polypeptide expressed in the plant by the process of cosuppression, in a manner similar to that discussed, for example, by Napoli et al. (Plant Cell 2:279-290, 1990) and de Carvalho Niebel et al. (Plant Cell 7:347-358, 1995).
Alternatively, regulation of polypeptide expression can be achieved by inserting appropriate sequences or subsequences (e.g. DNA or RNA) in ribozyme constructs (McIntyre C L, Manners J M, Transgenic Res., 5(4): 257-262, 1996). Ribozymes are synthetic RNA molecules that comprise a hybridizing region complementary to two regions, each of which comprises at least 5 contiguous nucleotides in a mRNA molecule encoded by one of the inventive polynucleotides. Ribozymes possess highly specific endonuclease activity, which autocatalytically cleaves the mRNA.
The DNA sequence of interest is operably linked to a polynucleotide promoter sequence of the present invention such that a host cell is able to transcribe an RNA from the promoter sequence linked to the DNA sequence of interest. The gene promoter sequence is generally positioned at the 5xe2x80x2 end of the DNA sequence to be transcribed. Use of a constitutive promoter, such as the ubiquitin polynucleotide promoter sequence of SEQ ID NO: 2 and 3, will affect transcription of the DNA sequence of interest in all parts of the transformed plant. Use of a tissue specific promoter, such as the leaf-specific promoter of SEQ ID NO: 9-11 or the root-specific promoter of SEQ ID NO: 13 and 14, will result in production of the desired sense or antisense RNA only in the tissue of interest. Temporally regulated promoters, such as the xylogenesis-specific promoter of SEQ ID NO: 5, can be employed to effect modulation of the rate of DNA transcription at a specific time, during development of a transformed plant. With DNA constructs employing inducible gene promoter sequences, the rate of DNA transcription can be modulated by external stimuli, such as light, heat, anaerobic stress, alteration in nutrient conditions and the like.
The inventive DNA constructs further comprise a gene termination sequence which is located 3xe2x80x2 to the DNA sequence of interest. A variety of gene termination sequences which may be usefully employed in the DNA constructs of the present invention are well known in the art. One example of such a gene termination sequence is the 3xe2x80x2 end of the Agrobacterium tumefaciens nopaline synthase gene. The gene termination sequence may be endogenous to the target plant or may be exogenous, provided the promoter is functional in the target plant. For example, the termination sequence may be from other plant species, plant viruses, bacterial plasmids and the like.
The DNA constructs of the present invention may also contain a selection marker that is effective in cells of the target organism, such as a plant, to allow for the detection of transformed cells containing the inventive construct. Such markers, which are well known in the art, typically confer resistance to one or more toxins. One example of such a marker is the NPTII gene whose expression results in resistance to kanamycin or hygromycin, antibiotics which are usually toxic to plant cells at a moderate concentration (Rogers et al. in Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press Inc., San Diego, Calif. (1988)). Transformed cells can thus be identified by their ability to grow in media containing the antibiotic in question. Alternatively, the presence of the desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern and Western blots.
Techniques for operatively linking the components of the inventive DNA constructs are well known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Sambrook et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). The DNA construct of the present invention may be linked to a vector having at least one replication system, for example E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the correctness of the manipulation determined.
The DNA constructs of the present invention may be used to transform a variety of target organisms including, but not limited to, plants. Plants which may be transformed using the inventive constructs include both monocotyledonous angiosperms (e.g. grasses, corn, grains, oat, wheat and barley) and dicotyledonous angiosperms (e.g. Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple), and Gymnosperms (e.g. Scots pine (Aronen, Finnish Forest Res. Papers, vol. 595, 1996), white spruce (Ellis et al., Biotechnology 11:94-92, 1993), and larch (Huang et al., In Vitro Cell 27:201-207, 1991)). In a preferred embodiment, the inventive DNA constructs are employed to transform woody plants, herein defined as a tree or shrub whose stem lives for a number of years and increases in diameter each year by the addition of woody tissue. Preferably the target plant is selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. Other species which may be usefully transformed with the DNA constructs of the present invention include, but are not limited to: pines such as Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellioti, Pinus jeffreyi, Pinus lambertiana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana; other gymnosperms, such as Abies amabilis, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnifica, Abies procera, Chamaecyparis lawsoniona, Chamaecyparis nootkatensis, Chamaecyparis thyoides, Huniperus virginiana, Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, Larix siberica, Libocedrus decurrens, Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea pungens, Picea rubens, Picea sitchensis, Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, Tsuga canadensis, Tsuga heterophylla, Tsuga mertensiana, Thuja occidentalis, Thuja plicata; and
Eucalypts, such as Eucalyptus alba, Eucalyptus bancroftii, Eucalyptus botyroides, Eucalyptus bridgesiana, Eucalyptus calophylla, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus coccifera, Eucalyptus curtisii, Eucalyptus dalrympleana, Eucalyptus deglupta, Eucalyptus delagatensis, Eucalyptus diversicolor, Eucalyptus dunnii, Eucalyptus ficifolia, Eucalyptus globulus, Eucalyptus gomphocephala, Eucalyptus gunnii, Eucalyptus henryi, Eucalyptus laevopinea, Eucalyptus macarthurii, Eucalyptus macrorhyncha, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus megacarpa, Eucalyptus melliodora, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus novaanglica, Eucalyptus obliqua, Eucalyptus obtusiflora, Eucalyptus oreades, Eucalyptus pauciflora, Eucalyptus polybractea, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus robusta, Eucalyptus rudis, Eucalyptus saligna, Eucalyptus sideroxylon, Eucalyptus stuartiana, Eucalyptus tereticornis, Eucalyptus torelliana, Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo and Eucalyptus youmanni; and hybrids of any of these species.
Techniques for stably incorporating DNA constructs into the genome of target plants are well known in the art and include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like. The choice of technique will depend upon the target plant to be transformed. For example, dicotyledonous plants and certain monocots and gymnosperms may be transformed by Agrobacterium Ti plasmid technology, as described, for example by Bevan (Nucl. Acid Res. 12:8711-8721, 1984). Targets for the introduction of the DNA constructs of the present invention include tissues, such as leaf tissue, dissociated cells, protoplasts, seeds, embryos, meristematic regions; cotyledons, hypocotyls, and the like. The preferred method for transforming eucalyptus and pine is a biolistic method using pollen (see, for example, Aronen 1996, Finish Forest Res. Papers vol. 595, 53pp) or easily regenerable embryonic tissues.
Once the cells are transformed, cells having the inventive DNA construct incorporated in their genome may be selected by means of a marker, such as the kanamycin resistance marker discussed above. Transgenic cells may then be cultured in an appropriate medium to regenerate whole plants, using techniques well known in the art. In the case of protoplasts, the cell wall is allowed to reform under appropriate osmotic conditions. In the case of seeds or embryos, an appropriate germination or callus initiation medium is employed. For explants, an appropriate regeneration medium is used. Regeneration of plants is well established for many species. For a review of regeneration of forest trees see Dunstan et al., Somatic embryogenesis in woody plants. In: Thorpe, T. A. ed., 1995: In Vitro Embryogenesis of Plants. Vol. 20 in Current Plant Science and Biotechnology in Agriculture, Chapter 12, pp. 471-540. Specific protocols for the regeneration of spruce are discussed by Roberts et al., (Somatic Embryogenesis of Spruce. In: Synseed. Applications of synthetic seed to crop improvement. Redenbaugh, K., ed. CRC Press, Chapter 23, pp. 427-449, 1993). Transformed plants having the desired phenotype may be selected using techniques well known in the art. The resulting transformed plants may be reproduced sexually or asexually, using methods well known in the art, to give successive generations of transgenic plants.
As discussed above, the production of RNA in target cells can be controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the DNA sequences incorporated into the genome of the target host. A target organism may be transformed with more than one DNA construct of the present invention, thereby modulating the activity of more than gene. Similarly, a DNA construct may be assembled containing more than one open reading frame coding for a polypeptide of interest or more than one untranslated region of a gene coding for such a polypeptide.
The isolated polynucleotides of the present invention may also be employed as probes to isolate polynucleotide promoter sequences from other species, using techniques well known to those of skill in the art, such as routinely employed DNA hybridization and PCR techniques.